This invention relates to the identification of sequence differences in alternative forms of rotavirus nonstructural protein 4 which result in significant changes in cytotoxicity.
Rotaviruses are recognized as the most important cause of severe viral gastroenteritis in humans and animals (Bibliography entry 1). Rotaviruses are non-enveloped, triple-layered particles with a genome consisting of 11 segments of double-stranded RNA. Group A rotaviruses are the major cause of rotaviral disease in humans and animals.
The prototypic Group A rotavirus, designated SA11, was discovered 10 years before human rotaviruses were identified. Malherbe et al. (2) described the isolation of a virus, simian agent 11 (SA11), from the rectum of a healthy vervet monkey and its cytopathic effects in vervet monkey kidney call cultures. The virus was distributed to Holmes et al. (Dept. Microbiology, University of Melbourne, Australia) (3), Estes et al. (Division of Molecular Virology, Baylor College of Medicine, Houston, Tex., USA) (4), and the American Type Culture Collection. Unfortunately, the precise passage history of each virus sample is not known.
The nonstructural protein 4, referred to as NSP4, is encoded by gene 10 of group A rotaviruses. Among the several putative roles of the multifunctional rotavirus nonstructural protein 4 (NSP4) is disease pathogenesis (5-11). That contention is based on several past studies of the NSP4 encoded by the prototypic rotavirus, strain SA11 (5-8).
The nucleotide sequence of gene 10 from the SA11 strain of rotavirus, a prototype of group A rotavirus, was reported by Both et al. in 1983 based on analysis of a plaque purified virus stock from Australia provided by Holmes et al. (12). The sequencing was performed by a combination of methods. Parts of the gene 10 were subcloned into bacteriophage M13 and sequenced by the Sanger method (13); other regions of the cDNA clone were sequenced by the method of Maxam and Gilbert (14).
Previous studies of NSP4 (5-8,11,12,15-23) are based upon a cDNA clone of this version of the NSP4 gene provided by Both et al. The original NSP4 sequence of the Australian strain has an asparagine at amino acid position 47 (NSP4 (Asn)) (12) in the deduced amino acid sequence.
NSP4 is a transmembrane protein with multiple functions. It acts as an intracellular receptor for subviral particles budding into the endoplasmic reticulum (ER) of infected cells (16,21). The expression of NSP4 results in increased intracellular calcium levels ([Ca2+]i) in Sf9 insect cells (5). Similar effects on intracellular calcium changes were observed with NSP4 from the OSU strain, a different group A rotavirus (9). A phospholipase C (PLC) mediated pathway is activated when NSP4 protein or synthetic peptide NSP4 114-135 is added exogenously to insect (6) or mammalian (8) cells.
A role for calcium in cytopathic effect (CPE) and cell death has been proposed for many pathological processes induced by virus (24). Increased [Ca2+]i levels have been linked to cytotoxicity in MA104 cells (monkey kidney cells; Biowhittaker Inc., Walkersville, Md.) infected with group A rotaviruses (25). It has been proposed that NSP4-induced cytotoxicity is due to an increase in intracellular calcium levels in infected cells (5). NSP4 has been found to be cytotoxic to mammalian cells (5,11). NSP4 has also been shown to function as an enterotoxin causing diarrhea in young mice (7). Antibodies against NSP4 provide passive protection against rotavirus in the same animal model (7).
There is a need to develop alternative forms of NSP4 which have reduced cytotoxicity, but which retain a conformation which approximates that of the prototypic rotavirus strain, such that they are still antigenic and immunogenic. Such antigenic and immunogenic forms are candidates for inclusion in antigenic compositions to protect against rotavirus disease.
Accordingly, it in an object of this invention to identify regions of NSP4 which are responsible for cytotoxicity and increased intracellular calcium levels.
It is a further object of this invention to identify and develop develop alternative forms of NSP4 which have reduced cytotoxicity, but which are still antigenic and immunogenic, because they retain a conformation which approximates that of the prototypic rotavirus strain.
As described herein, there is a histidine at amino acid position 47 of NSP4 in the SA11 ATCC rotavirus strain (NSP4 (His)) compared to an asparagine in the Australia rotavirus strain derived by Holmes et al. and sequenced by Both et al. (NSP4 (Asn)). This substituted form, NSP4 (His), retains the major functions of NSP4 (Asn), i.e. cytotoxicity and intracellular calcium changes, but it is more cytotoxic to cells.
To produce an alternative form of NSP4 which has reduced toxicity, while retaining its antigenicity and immunogenicity, the histidine or asparagine at amino acid position 47 of NSP4 is mutagenized to another amino acid. For example, the codon CAT encoding the histidine or the codon AAT encoding the asparagine at amino acid 47 is mutagenized to GAA, which encodes aspartic acid. Other attenuating mutations of this amino acid may also be used to reduce the toxicity.
As also described herein, there is a lysine at amino acid position 48 in both the SA11 ATCC and Australian rotavirus strains. An alteration of the lysine to glutamic acid increases the intracellular calcium levels and the toxicity caused by the virus. Thus, amino acid 48 is also identified as a position which contributes to viral toxicity. To produce an alternative form of NSP4 which has reduced toxicity, while retaining its antigenicity and immunogenicity, the lysine at amino acid position 48 is mutagenized to another amino acid other than glutamic acid.
Amino acids flanking amino acid positions 47 and 48 may also be mutagenized in a similar manner. Still further mutations in other regions of NSP4 may also be combined with the mutations at amino acid positions 47 and/or 48.
As further described herein, this invention also relates to an isolated and purified nucleic acid sequence comprising a nucleic acid sequence encoding: (a) rotavirus NSP4 protein in which the histidine or asparagine at amino acid position 47 is mutagenized to another amino acid to produce an alternative form of NSP4 which has reduced toxicity, while retaining its antigenicity and immunogenicity; (b) rotavirus NSP4 protein in which the lysine at amino acid position 48 is mutagenized to another amino acid other than glutamic acid to produce an alternative form of NSP4 which has reduced toxicity, while retaining its antigenicity and immunogenicity; or (c) rotavirus NSP4 protein in which the histidine or asparagine at amino acid position 47 is mutagenized to another amino acid, and in which the lysine at amino acid position 48 is mutagenized to another amino acid other than glutamic acid, to produce an alternative form of NSP4 which has reduced toxicity, while retaining its antigenicity and immunogenicity.
This invention also relates to the construction of a plasmid which expresses an alternative form of NSP4 as described above.
In order to obtain expression of the alternative form of NSP4, the isolated and purified nucleic acid sequence is first inserted into a suitable plasmid vector. A suitable host cell is then transformed, transfected or infected with the plasmid. In an embodiment of this invention, the host cell is an Sf9 cell. The host cell is then cultured under conditions which permit the expression of said alternative form of NSP4 by the host cell.
In another embodiment of this invention, the alternative forms of NSP4 protein are used to prepare antigenic compositions which elicit a protective immune response against rotavirus in a mammalian host, or which ameliorate diarrheal symptoms in such a host which has been infected with rotavirus. The antigenic composition may further comprise an adjuvant, diluent or carrier. Examples of such adjuvants include aluminum hydroxide, aluminum phosphate, MPL(trademark), Stimulon(trademark) QS-21, IL-12 and cholera toxin. The antigenic compositions are administered to a mammalian host in an immunogenic amount sufficient to protect the host against disease caused by rotavirus or to ameliorate diarrheal symptoms in such a host which has been infected with rotavirus.